The two major inhibitory synaptic transmitters, GABA and glycine, gate anion currents whose direction and magnitude are determined primarily by the distribution of chloride ions (Cl-) on either side of the neuronal membrane. This Cl- distribution s surprisingly difficult to determine. Each neuron seems to have a unique reversal potential for currents gated by GABAA receptors (EGABA), and recently EGABA has been shown to vary at different subcellular regions of the same neuron. These findings are hard to reconcile with the idea that one of two equilibrative cation-Cl cotransporters determine the Cl- distribution, because such a scheme links the Cl- distribution to the K and Na distributions, and should lead to an extremely uniform and predictable EGABA. ++ In the last grant cycle, we found a candidate explanation for the variability of EGABA: Cl is displaced by anionic - macromolecules in the cytoplasm and extracellular space. This is congruent with the concentration of impermeant anions in the cytoplasm, and the density of variably-sulfated glycosaminoglycans that largely comprise the matrix filling the extracellular space. The distribution of Cl- by charge displacement is analogous to the distribution of Styrofoam packing peanuts in a shipping box, where the shipped contents are the anionic macromolecules, the box is the neuronal membrane, and the peanuts are Cl-. Cl- distribution by displacement has interesting and testable predictions that we will begin to explore here. First, Cl- microdomains would be created if the distribution of anionic macromolecules is not uniform. Intracellular Cl microdomains would alter EGABA locally, while extracellular Cl domains would primarily affect -- the local GABAA conductance. Conceivably then, every GABAA synapse could have a unique reversal potential and conductance, which would permit the read-out of the enormous amount of information that could be stored in the distribution of Cl-displacing anionic macromolecules. The second prediction is that disruption of the extracellular matrix after brain injury, for example by the activation of matrix metalloproteases, would increase extracellular Cl-. Equilibrative co-transport of Cl-, cations, and water would then increase intracellular volume - a new mechanism for cytotoxic edema. We will test these predictions using perforated patch recordings and novel high-resolution Cl- reporting tools. These tools include transgenic mice with inducible expression of more sensitive ratiometric fluorescent Cl- fluorophores. We are also developing, as part of a collaborative BRAIN U01, fusions of these new Cl- fluorophores to the intracellular and extracellular faces of GABAA receptors. These fluorophores provide the sensitivity, stability and spatial resolution to rigorously test the Cl- microdomain hypothesis and the pathogenesis of cytotoxic edema using multi-photon and newly-developed very-long-term imaging technologies.